NOTCH signaling pathway is required for bovine early embryonic development

The NOTCH signaling pathway plays an important role in regulating various biological processes, including cell proliferation, lineage specification and apoptosis. Multiple components of the NOTCH pathway have been identified in mammalian preimplantation embryos. However, the precise role of NOTCH pathway in early embryonic development is poorly understood, especially in domestic animals. Here, we show that key transcripts of the NOTCH pathway exhibit a dynamic pattern throughout early embryonic development with an abundant level before embryonic genome activation in cattle. We also confirmed the presence of active NOTCH1 and RBPJ. By using pharmacological and RNAi tools, we demonstrated that the NOTCH pathway is required for the proper development of bovine early embryos. This functional consequence could be partly attributed to the major transcriptional mediator-RBPJ, whose deficiency also compromised the embryo quality. Indeed, we observed a significant increase of histone H3 serine 10 phosphorylation (pH3S10, a mitosis marker) positive blastomeres in not only NOTCH1 depleted embryos but RBPJ depleted ones. Importantly, RNA-seq analysis revealed that either NOTCH1 or RBPJ depletion triggers a reduction in H1FOO that encodes the oocyte-specific linker histone H1 variant. Interestingly, depleting H1FOO also results in detrimental effects on the developmental competence of early embryos, similar with NOTCH1 inhibition. Overall, our results reveal a crucial role for NOTCH pathway in regulating bovine preimplantation development, likely by controlling cell proliferation and maintaining H1FOO expression. Overall design: Late morula (D6) were harvested from nonspecific siRNA-injected control and NOTCH1 or RBPJ KD groups (n = 2; 15 embryos/group/replicate). Total RNA extraction was performed with a PicoPure RNA Isolation Kit based on the manufacturer's manual. Before RNA isolation, equal amounts of GFP and RFP mRNAs (2×106 copies) were added into each group as spike-in controls. mRNA separation was achieved using oligo(dT)25 beads. Sequencing libraries were constructed with NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs) based on the manufacturer's instructions.